Ca2+ enters into cells through a variety of Ca2+ channel (Cav) subtypes, as well as non-specific cationic channels, such as mechanically-gated channels. Upon entry, Ca2+ regulates the electrical and biochemical properties of hair cells (HCs) and spiral ganglion neurons (SGNs). Previous studies from our laboratory and others have made important new discoveries on the identification of the multiple Ca2+-mediated signaling in HCs and SGNs. However, the cellular mechanism of this Ca2+-mediated functions remains unclear. Additionally Ca2+ activates several outward channel currents, such as Ca2+-activated Cl- channels through mechanisms that still remains unknown. The overall goal of this proposal is to deploy innovative molecular biological, electrophysiological, and imaging techniques, many inspired from previous Ca2+ channel studies, for the discovery of fundamental and newly accessible arenas of Ca2+-mediated physiology in SGNs. This proposal drives three aims that address the HC and SGN Ca2+-mediated physiology, each with fundamental and therapeutic implications. The overall hypothesis is Ca2+ inflow into SGNs regulates distinct functions, ranging from short-term membrane excitability to long-term developmental processes. The Aims are: 1) To unequivocally resolve the functions of distinct subtypes of Ca2+ channels in SGNs. 2) To determine Ca2+-mediated mechanisms underlying the transformation of the features of pre- to post-hearing SGNs and finally 3) To determine the mechanisms underlying the reorganization of promiscuous innervation of HCs by SGNs prior to hearing onset. We predict that combined pre- and post-synaptic activities strengthen inner HC (IHC)-type 1-SGN synapse following initial indiscriminate contacts. The project will be conducted using different mouse models and their corresponding age-matched controls, as well as physiological, imaging and biochemical tools. Overall, this proposal will answer fundamental unknowns of Ca2+-mediated electrical and biochemical changes in HC and SGN physiology. Indeed, our findings are likely to reveal the mechanisms of pathological auditory phenomena, and in doing so, facilitate our efforts to design new therapeutic strategies. The discovery that developing SGNs are spontaneously active, and that this may alter SGN growth pattern as well as synapse formation are likely to have measurable impact in the design of new cochlear implants with increased precision and accuracy.